Project Summary Adoptive immunotherapy trials using genetically modified natural killer (NK) and T cells with redirected specificity against B cell malignancies have demonstrated therapeutic efficacy. Two early-phase clinical trials have been initiated at The University of Texas M. D. Anderson Cancer Center to evaluate the safety and efficacy of adoptive transfer of autologous and allogeneic T cells expressing a CD19-specific chimeric antigen receptor (CAR) into patients with advanced B-cell malignancies. These CD19-specific CAR+ T cells are selectively propagated in vitro in the presence of irradiated artificial antigen presenting cells (aAPC) expressing co-stimulatory molecules that sustain their proliferation to clinically-sufficient numbers in compliance with current good manufacturing practice (cGMP) for Phase I/II trials. This conventional culturing process requires 28 days to generate the required >1010 CAR+ T cells for each infusion. Reduction of the time required to generate clinically sufficient numbers in tissue culture will reduce manufacturing costs significantly and will yield a clinical product in which the CAR+ T cells have increased replicative potential and are less differentiated. To enhance the production process, we propose to develop novel co-culture protocol that utilizes magnetic co-levitation to increase the contact interface between CAR+ NK or T cells and the stimulating aAPCs to significantly improve the kinetics of numeric expansion of CAR+ lymphocytes with desired specificity and preservation of memory phenotype. Our long-term goal is to develop a cGMP-compliant protocol based on three-dimensional (3D) magnetic levitation co-cultures for the rapid and specific production of CAR+ T and NK cells to support ongoing immunotherapy trials. Our novel expansion method is based on the central hypothesis that increasing the number and duration of contact points between CAR+ T or NK cells and aAPCs will accelerate the proliferation of the targeting cells, yielding clinically sufficient number of cells in less time. In this Phase I SBIR proposal, we aim to develop and optimize 3D cell magnetic levitation co-culture conditions to improve the propagation of CAR+ T cells by magnetic nanoparticle labeling of aAPCs, NK, and T cells and co-culturing in 3D to maximize expansion of the genetically modified CD19-specific NK and T cells. Development of this expansion methodology will significantly impact the treatment, management, and cost of adoptive cell therapies of B cell malignancies.